Genome Sequence of Atyrau-5BJN(IL18), a Recombinant Lumpy Skin Disease Virus with Knockout of Virulence Genes

ABSTRACT Here, we present the coding sequence of the genome of the recombinant lumpy skin disease virus (LSDV) Atyrau-5BJN(IL18), obtained by knocking out four genes in the genome of a virulent field LSDV isolate. Genome sequencing confirmed the deletion of genes and the insertion of a foreign sequence in the viral genome.

L umpy skin disease virus (LSDV), genus Capripoxvirus of the family Poxviridae, is the causative agent of an infectious disease in cattle. Rapid diagnostics and vaccination are the basis for controlling the disease spread. The development of molecular biology methods has made it possible to obtain attenuated viruses by targeted mutagenesis of the viral genome (1).
Here, we present the coding sequence of the genome of the recombinant lumpy skin disease virus (LSDV) Atyrau-5BJN(IL18), obtained by knocking out four genes in the genome of a virulent field LSDV isolate. The primary virulent virus was isolated from an infected cow during an outbreak of lumpy skin disease in Kazakhstan in July 2016 (ProMED 20160722.4363497). Initially, four passages were performed in the MDBK cell line, and then four genes were knocked out by homologous recombination with transient dominant selection of recombinant viruses (2). We anticipate that the recombinant Atyrau-5BJN(IL18) will be harmless, immunogenic, and protect cattle during challenge with virulent virus (3).
The recombinant virus was produced in lamb testicle cell culture and purified by centrifugation in a sucrose density gradient (4). Genomic DNA was isolated using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol.
De novo genome assembly was performed using the program SPAdes v3.13 (8) with a k-mer length of 127 and the "-careful" option. As a result, a contig with a length of 145,167 bp was obtained; it was identified using blastn and the nucleotide collection (nt) database (9) as an LSDV genome.
To determine the depth of sequencing, the reads were mapped to the corresponding assemblies using the BWA-mem v0.7.17 algorithm (standard settings) (10), and the depths for each nucleotide and the median were calculated using SAMtools v1.15.1 (11). The sequencing depth was 255Â.
Inverted terminal repeat sequences were PCR amplified using AccuPrime Taq DNA polymerase, high fidelity, and determined by Sanger sequencing using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Austin, TX) on the 3130xl genetic analyzer (Applied Biosystems, Hitachi, Japan). The resulting contigs were manually combined into a sequence of 148,628 bp with an average G1C content of 25.87%, evenly distributed. About 600 bp were missing from both the 59 and 39 ends of the genome compared to the genome sequences present in GenBank.
Genome annotation was performed using GATU software relative to the LSDV isolate Kubash/KAZ/16 (GenBank accession number MN642592) (12). In addition to the targeted mutations introduced into the genome of the recombinant virus, three nucleotide substitutions were identified that would lead to amino acid substitutions (Table 1).
Genome sequencing confirmed the deletions of genes and the insertion of a foreign sequence in the viral genome (Table 1).
Data availability. The genome sequence of the lumpy skin disease virus Atyrau-5BJN (IL18) has been deposited at GenBank under accession number ON005067, and the raw data have been submitted to the SRA under BioProject accession number PRJNA825391.